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Microbial synthesis of vanillin from waste

Cite this: DOI: 10.1039/d1gc00931a
poly(ethylene terephthalate)†
Open Access Article. Published on 10 June 2021. Downloaded on 6/17/2021 5:08:43 AM.

Joanna C. Sadler and Stephen Wallace *

Received 15th March 2021,
Accepted 12th May 2021
DOI: 10.1039/d1gc00931a
rsc.li/greenchem
Poly(ethylene terephthalate) (PET) is an abundant and extremely useful material, with widespread appli-
cations across society. However, there is an urgent need to develop technologies to valorise post-consu-
mer PET waste to tackle plastic pollution and move towards a circular economy. Whilst PET degradation
and recycling technologies have been reported, examples focus on repurposing the resultant monomers
to produce more PET or other second-generation materials. Herein, we report a novel pathway in engin-
eered Escherichia coli for the direct upcycling of PET derived monomer terephthalic acid into the value-
added small molecule vanillin, a flavour compound ubiquitous in the food and cosmetic industries, and an
important bulk chemical. After process optimisation, 79% conversion to vanillin from TA was achieved, a
157-fold improvement over our initial conditions. Parameters such as temperature, cell permeabilisation
and in situ product removal were key to maximising vanillin titres. Finally, we demonstrate the conversion
of post-consumer PET from a plastic bottle into vanillin by coupling the pathway with enzyme-catalysed
PET hydrolysis. This work demonstrates the first biological upcycling of post-consumer plastic waste into
vanillin using an engineered microorganism.

Plastics have widespread and important applications across
society, yet poor management of these fossil fuel derived
resources is causing widespread pollution. The global plastic
waste crisis is now recognised as one of the most pressing
environmental issues facing our planet, prompting urgent
calls for new technologies to enable a circular plastics
economy.1–3 There is also a strong economic incentive, with
plastics losing 95% of their material value after a single use,
leading to an estimated $110 bn loss to the global economy
per annum.4 Therefore, new methods to degrade and valorise
plastic waste would have considerable economic as well as
environmental impact.
As such, the emerging field of plastic degradation and re-
cycling has attracted significant attention across the fields of
microbiology,5–7 biocatalysis and directed evolution,8 synthetic
biology1 and non-enzymatic catalysis.9–13 In particular, there
have been significant developments in enzymatic PET degra-
dation. The most promising enzymes discovered to date are
variants of PETase from Ideonella sakaiensis14 and leaf-branch
compost cutinase (LCC).15 Whilst PETase and its engineered
variants16–20 operate at ambient temperature (30–37 °C) to
Institute of Quantitative Biology, Biochemistry and Biotechnology, School of
Biological Sciences, University of Edinburgh, Roger Land Building, Alexander Crum
Brown Road, King’s Buildings, Edinburgh, EH9 3FF, UK.
E-mail: stephen.wallace@ed.ac.uk
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1gc00931a
release bis- and mono-(2-hydroxyethyl)terephthalate, the LCC
derived enzyme is thermostable with maximum productivity at
72 °C and exhibits the highest PET to terephthalate (TA) degra-
dation productivity reported to date (up to 16.7 g L−1 h−1).15
As increasingly efficient PET degradation methods are
developed,16,17,21–23 the recycling of PET monomers TA and
ethylene glycol has also been demonstrated. For example, TA
from LCC catalysed PET degradation has been used as a feed-
stock for production of second generation PET,15 and as a
building block for synthesis of value-added metal organic
frameworks.24 Post-consumer PET has also been pyrolyzed and
the TA produced used as a feedstock for bacterial production
of polyhydroxyalkanoates (PHAs),25 a promising class of bio-
plastics with a superior biodegradability profile to PET26,27
(Fig. 1a). Chemically hydrolysed PET has also been used as a
feedstock for protocatechuate (PC) derived small molecules
using microbial co-cultures.28 However, to date there have
been no examples of interfacing enzyme-catalysed PET degra-
dation with biological upcycling pathways. To this end, we
hypothesised that TA derived from PET waste could be
valorised into the value-added product vanillin via a 5-step
enzymatic pathway (Fig. 1b). Vanillin is the primary com-
ponent of the extract of vanilla beans and is responsible for
the characteristic taste and smell of vanilla. Consequently,
vanillin is widely used across the food and cosmetics indus-
tries, as well as in pharmaceutical synthesis and formulation,
herbicides, antifoaming agents and cleaning products, leading
to a global demand in excess of 37 000 tonnes in 2018.29 The

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Fig. 1 (a) Chemical and enzymatic degradation of PET has been used to recycle the monomer terephthalic acid for use in second-generation PET
and as a building block for value-added products such as metal–organic frameworks (MOFs), and as a feedstock for bioplastics production. (b) A
novel biosynthetic route to vanillin from plastic (PET) waste using engineered E. coli cells. (c) Biotechnological routes to vanillin from renewable or
waste feedstocks.
demand for vanillin is growing rapidly and is projected to
exceed 59 000 tonnes with a revenue forecast of $734 000 000
by 2025.29
As the demand for vanillin far exceeds the supply from
vanilla beans (‘natural vanillin’), methods for its synthesis
(‘synthetic vanillin’) have been widely investigated.
Biotechnological approaches are particularly attractive from a
sustainability perspective owing to the inherently mild reaction
conditions and use of renewable feedstocks.30 The most widely
employed of these is the production of vanillin from lignin
biomass,31 whilst methods for fermentation of vanillin using
microbial and fungal hosts from ferulic acid,32,33 glucose, gly-
cerol, L-tyrosine, xylose,34 curcumin,35 eugenol33 and isoeu-
genol36 have also been demonstrated (Fig. 1c). However,
although conversion of TA to vanillic acid is known,28 direct
access to the high value aldehyde vanillin from PET has not
been reported. Herein, we demonstrate a novel enzymatic
pathway using engineered Escherichia coli cells to synthesise
vanillin from TA. This process falls within the ‘biotechnologi-
cal vanillin’ sub-category of ‘synthetic vanillin’,30 namely vanil-
lin produced by microbial fermentation via a non-natural,
engineered pathway. After process optimisation, we obtain
conversions in excess of 79%. Finally, we show that the
pathway can be combined with LCC catalysed PET degradation
to obtain vanillin directly from post-consumer plastic waste at
ambient temperature and in aqueous conditions.
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Results and discussion
Construction of a novel enzymatic pathway for vanillin
production from TA
Our initial focus was on developing a novel in vivo enzymatic
pathway for the conversion of TA to the target compound vanil-
lin. Due to the inherent ability of E. coli to reduce aldehydes to
the corresponding alcohol,37 we chose E. coli MG1655 RARE
(reduced aromatic aldehyde reduction) as the host microorgan-
ism, which has previously been used for the biosynthesis of
vanillin from glucose.38 Inspired by the biodegradation and
metabolism of PET via PC by Ideonella sakaiensis,14 we hypoth-
esised that TA could be converted into vanillin by a de novo
pathway comprising: (i) terephthalate 1,2-dioxygenase
(TPADO), (ii) dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic
acid dehydrogenase (DCDDH), (iii) carboxylic acid reductase
(CAR) and (iv), catechol O-methyltransferase (COMT) (Fig. 2a).
It is noteworthy that CARs39 and O-MTs40 have both been
reported to accept PC, dihydroxybenzaldehyde (DHBAl) and
vanillic acid (VA) as substrates, such that the pathway may
proceed by two possible intermediates (VA or DHBAl) to
produce vanillin.
The pathway enzymes were assembled onto two plasmids,
named pVan1 and pVan2. pVan1 encodes TPADO from
Comamonas sp., a heterotrimer comprising subunits TphA1,
TphA2 and TphB2, and DCDDH, also from Comamonas sp.,
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Fig. 2 (a) Proposed enzymatic pathway for the conversion of PET to value added product vanillin. LCC: Leaf-branch compost cutinase; TPADO:
terephthalate 1,2-dioxygenase; DCDDH: 1,4-dicarboxylic acid dehydrogenase; O-MT: O-methyltransferase; CAR: carboxylic acid reductase; FAD:
flavin adenine dinucleotide; NAD(P)H: reduced nicotinamide adenine dinucleotide ( phosphate); SAM: S-adenosyl-L-methionine (b) design of
pathway enzyme expression constructs pVan1 and pVan2. (c) HPLC traces showing proof of concept for the pathway. Vanillin was only detected
when TA was added to cells expressing pVan1, pVan2 and pSfp.

which together catalyse the conversion of TA to PC using
atmospheric oxygen as the terminal oxidant. Both enzymes
have previously been shown to express in E. coli and show
activity towards TA and DCD, respectively.41–44 pVan2 encodes
a carboxylic acid reductase from Nocardia iowensis (NiCAR)
and a single point mutant of the soluble form of catechol
O-methyltransferase (S-COMT Y200L), from Rattus norvegicus
(Fig. 2b and Fig. S1†).39,40 This mutant was chosen for its high
stereoselectivity for methylation at the meta-position of PC and
DHBAl.40 NiCAR was selected for the reduction step as it has
previously been demonstrated to efficiently reduce both PC
and VA to the corresponding aldehydes.39 Additionally, cells
were co-transformed with a third plasmid encoding the phos-
phopantetheinyl transferase ( pSfp) from Bacillus subtilis,
which is necessary for post-translational modification of
NiCAR.45 For proof of concept, resuspended E. coli RARE cells
expressing pVan1, pVan2 and pSfp (E. coli RARE_pVanX) were
used in a screening scale biotransformation reaction for the
conversion of TA to vanillin. Whilst no vanillin was detected
from cells expressing only pVan1 or experiments lacking TA
substrate, cells expressing all three plasmids with added TA
(5 mM) gave a detectable amount of the target compound
vanillin (5 µM, <1% conversion) (Fig. 2c and Fig. S3–S5†), with
intermediates PC, DHBAl and vanillic acid also being detected
at concentrations of 18 µM, 10 µM and 2 µM, respectively
(Fig. S5†).
Process optimisation for maximum vanillin titres
Encouraged by these initial data, we sought to maximise vanil-
lin yields by optimisation of protein expression and whole cell
reaction conditions. A screen of protein expression media
showed M9 minimal media supplemented with casamino
acids (M9-CA) to give the highest combined levels of conver-
sion to vanillin and key intermediate PC (Fig. S5†).
Resuspending whole cells in fresh M9 media proved superior
to adding TA to expression cultures during the exponential
growth phase, giving a 4-fold increase in vanillin titres (77 µM
± 11 µM, Fig. 3a). This was hypothesised to be due to the
higher cell density and therefore biocatalyst loading of resus-
pended cells (OD600(ferm.) = 0.6–2; OD600(resusp.) = 20).
Examination of standard expression parameters such as induc-
tion OD, inducer concentration, time and temperature did not

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Fig. 3 Optimisation of vanillin pathway expression and biotransformation conditions. (a) Comparison between fermentation and resuspended
whole cells (conditions: 5 mM TA added to 10 mL fermentation reactions at time of induction, incubated at 30 °C for 24 hours). (b) Effect of cell
membrane permeabilisation (conditions: 5 mM TA, 30 °C, 24 hours). (c) Effect of addition of trace elements and benzyl alcohol (BnOH) to expression
medium (conditions: 5 mM TA, 30 °C, 24 hours). (e) Effect of in situ product removal (ISPR) (conditions: 1 mM TA, 22 °C, 24 hours). (e) Effect of bio-
transformation temperature (conditions: 5 mM TA, 24 hours). (f ) Time course of TA conversion under optimised conditions (1 mM TA, 22 °C, 20%v/v
oleyl alcohol (OA)). GTB: Glycerol tributyrate; TPGS: DL-α-tocopherol methoxypolyethylene glycol succinate; β-CD: β-cyclodextrin; PS-EDA: ethyle-
nediamine, polymer bound, 1% cross-linked (1%w/v loading); DVB-MEA: mercaptoethylamine, polymer bound, 1% cross-linked with divinylbenzene
(1%w/v loading). *P < 0.05, **P < 0.005, ***P < 0.0005 (Welch’s T-test).

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lead to significant improvement in vanillin yields. Likewise,
sulfur and iron containing expression medium additives such
as cysteine, ferrous sulfate and ferric ammonium citrate,
which were hypothesised to increase expression levels of [2Fe–
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2S] containing TPADO, did not give a significant increase in
TA conversion levels (Fig. S6†).43,46 However, the addition of
trace elements to the growth media led to a 1.5-fold increase
in PC titres (Fig. 3c). This was hypothesised to improve TPADO
and DCDDH activity, which are Fe2+ and Zn2+ dependent,
respectively.41,43,44 Interestingly, the addition of benzyl alcohol
(BnOH) to the expression culture prior to induction led to a
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2-fold increase in PC levels even in the absence of trace
elements. Benzyl alcohol is an osmolyte that has been reported
to induce expression of endogenous chaperones in E. coli,
leading to increased soluble expression of heterologous
genes.47 Accordingly, analysis of protein expression by
SDS-PAGE showed more soluble protein expression in the pres-
ence of BnOH or trace metals, whilst adding both BnOH and
trace metals to the expression culture did not give further
increase in either soluble protein expression or vanillin yields
(Fig. 3c and Fig. S7†).
Next, we investigated the effect of the whole cell biotrans-
formation conditions on conversion of TA to vanillin and
pathway intermediates. As TPADO is O2 dependant, we hypoth-
esised that increasing reaction headspace would increase con-
version of TA to PC. This was confirmed by observing a 65-fold
improvement in vanillin titres (5 µM ± 3 µM to 327 µM ±
15 µM) when the headspace to reaction volume ratio was
increased from 1 : 5 to 1 : 99 (Fig. S8†).
Further improvement in TA conversion was accomplished
by increasing E. coli cell membrane permeability to TA. Whilst
an influx transporter for TA in Pseudomonas sp. has been
reported,48 E. coli is not known to express a transporter
capable of importing TA to the cytosol. Therefore, cell mem-
brane permeabilization strategies were investigated. In particu-
lar, n-butanol (n-BuOH) has been shown to improve cell mem-
brane permeability towards small molecules49,50 and was
therefore hypothesised to improve TA conversion by increasing
substrate concentration in the cell interior. Whilst 0.1%v/v had
little effect on conversion, the addition of 1%v/v n-BuOH to
the biotransformation buffer resulted in a 3-fold increase in
cumulative conversion of TA to vanillin and pathway inter-
mediates (Fig. 3b). Conversely, attempts to improve membrane
permeability through freeze/thaw cycles or the addition of lyso-
zyme to the reaction buffer did not lead to increased vanillin
concentrations. Experiments using lysed E. coli RARE_pVanX
cells and experiments using co-cultures of E. coli RARE_pVan1
and E. coli RARE_pVan2_pSfp gave negligible (<1%) levels of
vanillin, indicating that co-localisation of the pathway enzymes
within a single cell is advantageous in this system. Finally, the
addition of L-Met (10 mM) resulted in a 2-fold increase in
vanillin titres (Fig. S9†). This aligns with previous reports of
in vivo pathways employing S-adenosylmethionine (SAM)
dependant methyltransferases (MTs), which note supplemen-
tation of reaction buffer with L-Met as being vital to achieve
maximum product titres.51
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The doubly charged nature of terephthalate at neutral pH
was also theorised to impede diffusion of TA across the cell
membrane and hence the effect of biotransformation buffer
pH was investigated. As predicted, a sharp decrease in TA to
PC conversion from pH6 to 8 was observed. Upon screening
conditions <pH7, pH5.5 was found to be optimum to balance
maximum TA diffusion into the cell whilst minimising acid
induced stress to the cell (Fig. S10†).
Cell density of resuspended E. coli RARE_pVanX was also
studied. Whilst there was minimal effect on product titres and
distribution between OD600 = 10–60, a significant increase in
vanillin concentration (350 µM ± 62 µM to 480 µM ± 36 µM)
was obtained at OD600 = 80, with a switch from PC to vanillin
being the predominant product (Fig. S11†).
Biotransformation temperature also had a dramatic effect
on vanillin yields (Fig. 3e). Decreasing the reaction tempera-
ture from 30 °C to 22 °C gave a 5-fold improvement in vanillin
yields (577 µM ± 22 µM compared to 117 µM ± 40 µM at
30 °C). Interestingly, the ratio of pathway intermediates
formed also changed upon lowering the reaction temperature.
Vanillic acid (VA) was produced at 30 °C (20 µM ± 8 µM),
whereas VA titres from lower temperature reactions were negli-
gible. This indicates that at biotransformation temperatures of
22 °C and lower, the pathway proceeds via NiCAR mediated
reduction of PC followed by S-COMT mediated methylation to
give vanillin, whereas at 30 °C the alternative pathway via vanil-
lic acid also operates. Decreasing the temperature to 16 °C
gave no further improvement in product conversion.
Finally, we hypothesised that in situ product removal (ISPR)
could improve vanillin yields through mitigating the toxicity
of vanillin to E. coli (Fig. S12†) and increasing flux towards
vanillin, the most hydrophobic molecule in the pathway
(log Pvanillin = 1.2).52 Three ISPR strategies were investigated:
(i) organic solvent overlays,53 (ii) product entrapment in bio-
compatible micelles54 or β-cyclodextrin55 and (iii) product trap-
ping via reversible nucleophilic addition to the aldehyde
moiety of DHBAl and vanillin.56,57 Whilst the product trapping
reagents did not improve product titres, all the solvent overlays
and product entrapment ISPR reagents increased vanillin
yields relative to the control experiment (Fig. 3d). Out of these,
oleyl alcohol (OA) and vitamin E derived biocompatible
micelles TPGS-750-M were selected for further study based on
initial data showing the highest vanillin concentrations and
lowest levels of the intermediate DHBAl.
The biotransformation of TA to vanillin in the presence of
no ISPR reagent, 20%v/v OA, or 2%w/v TPGS-750-M was
carried out with varying TA concentrations to investigate
whether ISPR enabled higher vanillin titres. These data
showed OA to be the superior ISPR reagent, giving maximum
vanillin titres of 744 µM ± 100 µM from 1 mM TA. The pres-
ence of OA also led to increased conversion of PC to DHBAl,
however there was no significant increase in vanillin titres
with increased TA concentration, indicating the bottleneck in
the pathway to be the final COMT mediated methylation step
(Fig. S13†). Strategies to alleviate this bottleneck, such as
screening a library of alternative O-MTs and upregulating
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expression of SAH degradation enzymes,58,59 will be the focus
of further study and development of this pathway. Finally, a
time course experiment under the optimised conditions with
an oleyl alcohol overlay showed maximum vanillin titres to be
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obtained after 16 hours, with sequential formation and con-
sumption of PC and DHBAl also being observed (Fig. 3f ). This
led to the final optimised biotransformation conditions for TA
to vanillin conversion: namely E. coli RARE_pVanX cells resus-
pended in M9-glucose supplemented with L-Met and nBuOH,
pH 5.5, incubated with TA for 24 hours at room temperature
with an oleyl alcohol overlay. Product formation under the
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optimised conditions was confirmed upon scaling up the bio-
transformation to 40 mL scale and analysing the reaction pro-
ducts by NMR spectroscopy (Fig. S14†).
Upcycling of post-consumer PET waste into vanillin
Finally, we set out to demonstrate the application of the TA to
vanillin pathway in valorisation of post-consumer plastic
waste. We selected the thermostable enzyme LCC WCCG15
(hereafter referred to as LCC) as a biocatalyst to aid hydrolysis
of PET into TA. Unlike PETase from Ideonella sakaiensis, LCC
releases TA directly and does not require an additional enzyme
to hydrolyse mono-2-hydroxyethyl terephthalate (MHET) for
release of TA. PET from a post-consumer plastic bottle was
treated with semi-purified LCC at 72 °C (Fig. 4a). The reaction
was cooled to room temperature and freshly prepared E. coli
RARE_pVanX and a biotransformation buffer concentrate were
Fig. 4 Conversion of a post-consumer PET bottle into vanillin. (a)
Overview of the one-pot, two-step process to convert PET into value-
added product vanillin. E. coli RARE_pVanX refers to E. coli RARE expres-
sing plasmids pVan1, pVan2 and pSfp. (b) Data showing production of
vanillin only in the presence of cells expressing the vanillin pathway
enzymes and the feedstock PET.
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added, and reactions were analysed after 24 hours.
Gratifyingly, without any process optimisation, vanillin was
detected when all pathway components were present (68 µM,
Fig. 4b). Vanillin was not detected in control experiments
lacking PET or cells expressing the pathway enzymes. Low
levels of vanillin were detected in experiments lacking LCC,
which is hypothesised to be due to background PET hydrolysis
in LCC reaction buffer ( pH10) in the absence of LCC
(Fig. S15†). The addition of an oleyl alcohol overlay did not
lead to a significant increase in vanillin titres, which was
hypothesised to be due to the lower TA concentrations from
PET degradation (300–400 µM).
Conclusions
Low cost and low intensity technologies to valorise post-consu-
mer plastic waste are urgently required to tackle the plastic
waste crisis and enable a circular economy. We have addressed
this by engineering a novel biosynthetic pathway in the labora-
tory bacterium E. coli to convert plastic derived monomer ter-
ephthalic acid directly into the value-added molecule vanillin
using a single engineered microorganism. The reaction is
mild, uses a whole-cell catalyst produced from renewable feed-
stocks and occurs under ambient conditions (room tempera-
ture, pH5.5–7), in aqueous media, requires no additional
cofactors or reagents and generates no hazardous waste.
Maximum vanillin titres of 785 µM (119 mg L−1, 79% conver-
sion) were achieved after extensive process optimisation
studies, a 157-fold improvement over titres from pre-optimi-
sation experiments. Key to this high conversion was biotrans-
formation temperature, cell permeabilisation and use of ISPR
to increase flux towards vanillin. Moreover, we have demon-
strated that this pathway can utilise TA derived directly from
post-consumer plastic waste in a one-pot bioprocess – the first
example of biological upcycling of waste PET into a single
value-added small molecule. Future studies will focus on
intensifying this process through further strain engineering,
process optimisation and extension of the pathway to other
metabolites. Fundamentally, this work substantiates the phil-
osophy that post-consumer plastic may be viewed not as a
waste product, but rather as a carbon resource and feedstock
to produce high value and industrially relevant materials and
small molecules.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
J. C. S. acknowledges a Discovery Fellowship from BBSRC (BB/
S010629/1) and S. W. acknowledges a Future Leaders
Fellowship from UKRI (MR/S033882/1).
This journal is © The Royal Society of Chemistry 2021

Green Chemistry
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